Previously we showed that the 5-HT present in the microvessels is not only derived from serontonergic nerve endings but it is also formed in the endothelium where its level is most probably controlled by prostaglandin. The continuous investigation comprised daily monitoring of 5-HT and 5-HIAA content in the feeding solution incubated with the cerebrovascular endothelium (experimental) and without these cells (control), permitting a direct determination of 5-HT metabolism and indirect assessment of endothelial 5-HT synthesis. The daily concentrations of each substance was assayed by HPLC. The findings of double or tripled 5-HIAA level with an absent or significantly lower content of 5-HT in the experimental than control medium could not be entirely derived from the available 5-HT in the feeding solution since the 5-HIAA concentration exceeded even the amount of 5-HT apparently taken up and metabolized by the cells (after 72-96 hrs of incubation). Thus, the accumulated 5-HIAA in the experimental medium most likely was derived from newly synthetized 5-HT in these cultures.